1. Field of the Invention
This invention relates to estrogen receptors and ligands for them, and in particular to crystalline estrogen xcex2 receptor (ERxcex2) and to methods of identifying ligands utilizing crystalline ERxcex2.
2. Brief Description of the Art
The thyroid hormone receptor (TR) is known and its three-dimensional structure, and hence its ligand binding domain, has been determined. Knowledge of the three-dimensional structure has enabled a better understanding of the modes of ligand binding and the determination of the optimum conformation of ligand to bind to the receptor. It is generally believed in the art that the TR structure also provides a guide to the design of ER ligands.
Estrogen steroid hormone and thus the estrogen receptor (ER) is a member of the steroid hormone receptor family. Its primary natural ligand is estradiol (E2). However, it is known that a large number of structurally diverse non-steroidal compounds such as raloxifene, centchroman, coumestrol, diethylstilbesterol, esculin, tamoxifen, zearalenone, and zindoxifen also bind to the estrogen receptor (FIG. 4). The majority of these non-steroidal estrogen receptor ligands contain 2-4 carboxylic, aromatic, and/or heterocyclic rings connected by a 1-3 atom chain. One or more of the rings may be fused with the central atom chain or with each other.
It has been proposed that the receptor possesses a multifunctional modular structure potentially having discrete domains for DNA binding, ligand binding, and transactivation. The ligand binding domain (LBD) has been designated domain E and is the largest domain of the estrogen receptor. The ligand binding domain includes a ligand recognition site and regions for receptor dimerization interation with heat shock proteins, nuclear localization and ligand dependent transactivation.
A review of the structure and functioning of the estrogen receptor is provided in an article by Katzenellenbogen, J. et al., Steroids, (1997) 62(3):268-303.
It is known that compounds which bind to the estrogen receptor are potentially useful in the treatment of a wide range of disease states. These include estrogen agonists for treatment of disease linked to estrogen deficiency (e.g. osteoporosis, cardiovascular and neurodegenerative diseases in post menopausal women) and estrogen antagonists for treatment of breast and uterine cancer. Furthermore, it is known that certain ligands such as tamoxifen display mixed agonist/antagonist action (that is they are either estrogen agonists, estrogen antagonists, or a partial estrogen antagonists when binding to the estrogen receptors of different tissues) and such compounds may simultaneously prevent bone loss and reduce the risk of breast cancer. It is further known that benzothiophenes are usable as agonists or antagonists to steroid hormones, and that it is possible to modify their binding mechanics, for example the binding affinity, by changing the substituent groups at various positions on the molecule. Therefore, it would be desirable to be able to design ligands which are recognizable by and able to bind to the estrogen receptor. Additionally, it would be desirable to know the three dimensional structure of the estrogen receptor. Such knowledge would be useful for the design of compounds intended to bind to the estrogen receptor. The present inventors have been able to produce an estrogen receptor crystal and to determine from that the three dimensional structure of the estrogen receptor. Unexpectedly, the thus determined ER structure reveals that the TR structure does not provide a good model for binding of ligands to ER.
Our copending patent application No. PCT/GB98/01708 discloses inter alia, the crystal co-ordinates of crystalline estrogen receptor alpha (ERxcex1).
We have now succeeded in crystallizing ERxcex2 bound to a ligand which is an antagonist or partial agonist and determining its crystallographic co-ordinates. Therefore, in a first aspect the present invention provides a crystal comprising at least 150 amino acid residues of the ERxcex2 ligand binding domain complexed with a ligand which is an antagonist or a partial agonist.
In a second aspect, the present invention provides ligands, particularly synthetic ligands, of ERxcex2 identified by use of such a crystal.
In a third aspect of the invention, methods for designing ligands which will bind to ERxcex2 are provided. Such methods use three dimensional models based on the crystals of the estrogen receptor ligand complex. Generally, such methods comprise determining compounds which are likely to bind to the receptor based on their three dimensional shape compared to that of the ERxcex2 and in particular the ligand binding domain of the ERxcex2. Preferably, those compounds have a structure which is complementary to that of the ERxcex2. Such methods comprise the steps of determining which amino acid or amino acids of the ligand binding domain of the ERxcex2 interacts with the binding ligand, and selecting compounds or modifying existing compounds, to improve the interaction. Preferably, improvements in the interaction are manifested as increases in the binding affinity but may also include increases receptor selectivity and/or modulation of efficacy.
Preferably the ligands bind to the ERxcex2 with a high binding affinity, for example within the range of 20-2000 pmol.
The ligands may bind tightly to the ERxcex2 yet not up-regulate gene expression thereby inhibiting the action of estradiol and estradiol mimetics. Thus, the invention also provides a method of inhibiting the activity of estradiol or estradiol mimetics by providing ligands which bind to ERxcex2 with high affinity, blocking the activity of the estrogens. Alternatively, binding of the ligand to the ERxcex2 may cause conformational changes to the ERxcex2 inhibiting further binding thereto. The invention further provides a method of inhibiting estradiol activity in an animal, the method comprising administering to the animal a ligand which binds to at least the LBD, of the ERxcex2 with high affinity and blocks binding of further ligands to at least the LBD of the ERxcex2. Such ligands are useful in, for example, the treatment of estrogen receptor mediated diseases in females.